Exon14 Skipping Research Articles

In vitro effectiveness of CB469, a MET tyrosine kinase inhibitor in MET-activated cancer cells

Gene alterations in receptor tyrosine kinases can result in oncogenic driver mutation in non-small cell lung cancer (NSCLC) including in genes like EGFR, ALK and MET. MET amplifications and MET exon14 skipping are the primary genetic changes in MET-altered cancers. Acquired MET mutations mediate resistance to clinical MET-targeted therapy in NSCLC. MET kinase domain secondary mutations (D1228X, Y1230X) confer resistance to type I MET tyrosine kinase inhibitors (TKIs) in METexon14-altered or MET amplified NSCLC. Here, we investigated the preclinical activity of a novel MET inhibitor, CB469, with cell growth, signaling pathway and colony formation. We confirmed that CB469 inhibited the activity of MET wild and secondary mutant kinases, D1228N and Y1230H, as a type II inhibitor. CB469 also inhibited cell growth and cell signaling proteins in MET-activated or MET exon14 skipping-mutated cancer cell lines and NIH/3T3 cells expressing an engineered MET mutant. CB469 exhibited the inhibitory efficacy comparable with that of capmatinib in migration of EBC-1(METwt) and Hs746T(METΔex14) cells. Finally, CB469 showed selective and potent inhibition in MET-activated cancer cells among MET TKIs leading to enhanced selectivity for MET-mutant versus wild type MET with inhibition of cell growth in NIH/3T3 cells expressing an engineered MET mutant variant.

Early-Stage Non-Small Cell Lung Cancer: Prevalence of Actionable Alterations in a Monocentric Consecutive Cohort.

Early-stage (ES) non-small cell lung cancer (NSCLC) is diagnosed in about 30% of cases. The preferred treatment is surgery, but a significant proportion of patients experience recurrence. Neoadjuvant and adjuvant chemotherapy has a limited clinical benefit. EGFR tyrosine kinase inhibitors and immunotherapy have recently opened new therapeutic scenarios. However, only a few data are available about the ES-NSCLC molecular landscape and the impact of oncogene addiction on therapy definition. Here, we determined the prevalence of the main lung cancer driver alterations in a monocentric consecutive cohort. Molecular analysis was performed on 1122 cases, including 368 ES and 754 advanced NSCLC. The prevalence of actionable alterations was similar between early and advanced stages. ES-NSCLC was significantly enriched for MET exon-14 skipping alterations and presented a lower prevalence of BRAF p.(V600E) mutation. PD-L1 expression levels, evaluated according to actionable alterations, were higher in advanced than early tumors harboring EGFR, KRAS, MET alterations and gene fusions. Taken together, these results confirm the value of biomarker testing in ES-NSCLC. Although approved targeted therapies for ES-NSCLC are still limited, the identification of actionable alterations could improve patients' selection for immunotherapy, favoring the enrollment in clinical trials and allowing a faster treatment start at disease recurrence.

Abstract 2116: Telisotuzumab vedotin (Teliso-V)-mediated c-Met internalization, endolysosomal trafficking, and potent cytotoxic activity are unaffected by MET exon-14 skipping mutation

Abstract Background: Telisotuzumab vedotin (Teliso-V) is an antibody-drug conjugate (ADC) composed of a c-Met (MET protein) antibody coupled to the microtubule inhibitor monomethyl auristatin E (MMAE). Teliso-V was granted FDA Breakthrough Therapy Designation for advanced/metastatic EGFR WT, NSQ NSCLC with high c-MET overexpression. For MMAE drug delivery, Teliso-V must first undergo receptor internalization and endolysosomal (EL) trafficking. Hepatocyte growth factor (HGF)-dependent MET EL trafficking and degradation depend on the E3 ubiquitin ligase Casitas B-lineage lymphoma (CBL). METex14 skipping and CBL-binding site mutations, which delete the binding site for CBL, occur in ~ 3% of NSCLC. The role of CBL in Teliso-V-MMAE delivery and activity was assessed in this work. Methods: WT-human c-Met, c-Met-Y1003F/N (CBL binding site mutations in MET), and c-Met-ex14∆ (47 aa deletion to mimic ex14 skipping) or CBL-L620f, G807*, and W802* (CBL inactivating mutants) were expressed in NIH3T3, Ba/F3 cells, or A549 tumor cells using retroviral transduction. Genetically modified cells were then used to define the role of CBL in c-Met surface expression, intracellular signaling, EL trafficking (using pHrodo Red-labeled Teliso-V), and Teliso-V sensitivity in 2D/3D cell cultures. Results: c-Met expression was required for Teliso-V cytotoxicity and intracellular payload delivery in all cell lines. While minimal changes in cell surface c-Met levels were observed, CBL binding mutants had increased phosphorylated c-Met in the absence of HGF, and ex14∆-MET and CBL binding site mutants showed increased sensitivity to small molecule c-Met inhibitors. Compared to WT c-Met, ex14∆ and CBL-binding mutants showed similar EL trafficking and no change in sensitivity to Teliso-V. Co-treatment with a pan-CBL inhibitor also failed to alter Teliso-V EL trafficking and anti-tumor activity. Like most ADCs, EL trafficking was dependent on a dynamin-mediated process with reduced EL trafficking by co-treatment with Dyngo4a. To further understand the role of CBL in Teliso-V sensitivity, we expressed CBL inactivating mutations in A549 tumor cells. While the L602F and W802* mutants increased spheroid size, as well as activated p-MET, there was no change in sensitivity to Teliso-V. Conclusions: Loss of CBL-binding sites on c-Met via ex14 deletion or Y1003F/N leads to increased MET-targeted TKI sensitivity and increased phospho-c-Met levels but does not impact Teliso-V sensitivity or EL trafficking. Our results indicate that Teliso-V traffics to the EL via a dynamin-mediated process. Therefore, Teliso-V delivers MMAE through a robust ADC-mediated EL-dependent mechanism that is independent of CBL, suggesting that high c-Met expression in Ex14skipping or CBL binding site mutant NSCLC patients should be further investigated for response to Teliso-V. Citation Format: Izhar Batth, Ronald Pandoy, Shamim Khaja, Dolonchampa Maji, Tamar Uziel, Athan Vasilopoulos, Peter Ansell, PK Epling-Burnette. Telisotuzumab vedotin (Teliso-V)-mediated c-Met internalization, endolysosomal trafficking, and potent cytotoxic activity are unaffected by MET exon-14 skipping mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2116.

Non-Small Cell Lung Cancer Testing on Reference Specimens: An Italian Multicenter Experience.

Biomarker testing is mandatory for the clinical management of patients with advanced non-small cell lung cancer (NSCLC). Myriads of technical platforms are now available for biomarker analysis with differences in terms of multiplexing capability, analytical sensitivity, and turnaround time (TAT). We evaluated the technical performance of the diagnostic workflows of 24 representative Italian institutions performing molecular tests on a series of artificial reference specimens built to mimic routine diagnostic samples. Sample sets of eight slides from cell blocks of artificial reference specimens harboring exon19 EGFR (epidermal growth factor receptor) p.E746_AT50del, exon2 KRAS (Kirsten rat sarcoma viral oncogene homologue) p.G12C, ROS1 (c-ros oncogene1)-unknown gene fusion, and MET (MET proto-oncogene, receptor tyrosine kinase) Δ exon14 skipping were distributed to each participating institution. Two independent cell block specimens were validated by the University of Naples FedericoII before shipment. Methodological and molecular data from reference specimens were annotated. Overall, a median DNA concentration of 3.3ng/µL (range 0.1-10.0ng/µL) and 13.4ng/µL (range 2.0-45.8ng/µL) were obtained with automated and manual technical procedures, respectively. RNA concentrations of 5.7ng/µL (range 0.2-11.9ng/µL) and 9.3ng/µL (range 0.5-18.0ng/µL) were also detected. KRAS exon2 p.G12C, EGFR exon19 p.E736_A750del hotspot mutations, and ROS1 aberrant transcripts were identified in all tested cases, whereas 15 out of 16 (93.7%) centers detected MET exon14 skipping mutation. Optimized technical workflows are crucial in the decision-making strategy of patients with NSCLC. Artificial reference specimens enable optimization of diagnostic workflows for predictive molecular analysis in routine clinical practice.

Abstract C152: Preclinical and emerging Phase 1 study data indicates that novel deuterated MET kinase inhibitor DO-2 mitigates the side effects seen with current approved MET kinase inhibitors: Preventing deleterious ‘de-hinging’ to improve tolerability

Abstract Introduction. MNNG HOS transforming gene (MET) kinase inhibitors have demonstrated clinical activity in patients with non-small cell lung cancer (NSCLC) harboring exon14 (METex14) skipping mutations, validating this as an actionable drug target. High daily doses (500-800mg daily) are needed to reach sufficient plasma exposure of active drug to achieve sufficient target engagement to drive efficacy. However, these high doses are associated with toxicities that frequently lead to dose interruptions and reductions which negatively impact efficacy. Background. The 2nd generation of selective Type 1b MET inhibitors (capmatinib, tepotinib, savolitinib), with more soluble metabolites than the 1st generation inhibitors (SGX-523, JNJ-38877605, PF-04217903), have shown efficacy in the clinic. However, they still carry the metabolic liability of the 1st generation agents translating into high levels of circulating metabolites that are not active against the MET kinase, necessitating high daily doses to achieve sufficient active drug exposures. These inactive ‘de-hinged’ metabolites (which are unable to interact with a key recognition site located in the ‘hinge region’ of the MET kinase) remain in the circulation, leading to ‘off-target’ toxicities which are not linked to efficacy, but often necessitates dose reductions and/or-interruptions. Experimental procedures. Based on a detailed understanding of the metabolic liabilities of type 1b MET kinase inhibitors and using site specific deuteration of the key metabolic hotspot, we have used the Kinetic Deuterium Isotope Effect (KDIE) to overcome a metabolic liability that negatively impacts current agents. Summary of the new, unpublished data. The resulting deuterated compound DO-2, is well tolerated and highly potent in pre-clinical xenograft studies (MED ~0.2mg/kg: MTD >50mg/kg), brain penetrant (Kp, uu, brain ; 0.3 in rats), with low once daily doses (3mg/kg) causing complete and sustained cures of all animals (8/8) carrying well established ~300mm3 MET exon14 xenografts. IND enabling toxicology studies in rabbit (most relevant species for metabolism) have shown that the renal effects of the predecessor JNJ-38877605 were no longer apparent with DO-2 at efficacious exposure levels. Based on the pre-clinical efficacy and toxicology data, DO-2 has entered the clinic in a Phase 1 study (NCT05752552), where early data indicates that the improved preclinical profile translates into the clinic with increased active drug exposure when compared to the non-deuterated JNJ-38877605. Conclusion. Preclinical data and emerging clinical data from highly MET inhibitor sensitive MET exon14 tumours as well as in other less sensitive MET driven tumors indicate that DO-2 has the potential to mitigate the side effects seen with current approved MET kinase inhibitors, leading to better tolerability and lower inactive metabolite levels and at lower daily dose levels. Supporting preclinical data and emerging clinical data will be presented. Citation Format: Timothy P.S. Perera, Laurence Mevellec, Hans Prenen, Bernd Dekeyser, Debbie Robbrecht, Sander Bins, Barend Sikkema, Peter de Bruijn, Jean-Pascal Machiels, Rachel Galot, Damien Briol, Richard Knight, Irena Loryan, Yang Hu, Florence Wastelin, Jaap Verweij. Preclinical and emerging Phase 1 study data indicates that novel deuterated MET kinase inhibitor DO-2 mitigates the side effects seen with current approved MET kinase inhibitors: Preventing deleterious ‘de-hinging’ to improve tolerability [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C152.

Real-World Treatment Outcomes of MET Exon14 Skipping in Non-small Cell Lung Cancer: GFPC 03-18 Study.

MET-targeted tyrosine kinase inhibitors (TKIs) demonstrated efficacy in advanced non-small cell lung cancer (aNSCLC) with MET exon14 skipping mutations (METexon14); yet, data on the management of these patients in clinical practice is sparse. The aim of this study was to describe the management of METexon14 aNSCLC patients. This real-life, retrospective study analyzed the management of METexon14 aNSCLC. The primary endpoint was the median overall survival (mOS). Secondary endpoints were to assess investigator-progression-free survival (PFS) and mOS in different subgroups: patients treated with (a) crizotinib, regardless of treatment line; (b) anti-MET TKIs (crizotinib, tepotinib, capmatinib); and (c) immunotherapy. A total of 118 patients were included between December 2015 and January 1, 2020 in 13 centers. Median age was 73 years, 62.7% were female, 83.9% had adenocarcinoma, 92.4% at stage IV, and 27% had more than three metastatic sites. The majority of the patients (106, 89.8%) received at least one systemic treatment; 73% received at least one anti-MET TKI: crizotinib (68.6%), tepotinib (16%), capmatinib (10%). Only 10% received two anti-MET TKIs in their treatment sequences. With a median follow-up of 16 months (95% CI 13.6-29.7), mOS was 27.1 months (95% CI 18-31.4). There was no significant difference between mOS of patients treated and never treated with crizotinib, 19.7 (95% CI 13.6-29.7) and 28 (95% CI 16.4-NR) months, respectively (p=0.16); mOS of the TKI cohort and of the TKI-naïve patient cohort were 27.1 (95% CI 18-29.7) and 35.6 (95% CI 8.6-NR) months respectively, with no significant difference (p=0.7). In this real-life study, there was no evidence of benefit in mOS with anti-MET TKIs.

A comparative analysis of next-generation sequencing based on plasma cfDNA and tumor tissue DNA in patients with advanced lung cancer.

e21021 Background: Although liquid biopsy based on plasma cell-free DNA (cfDNA) provides a non-invasive detection method for cancer patients, tissue biopsy is still the gold standard for molecular subtyping. The concordance of these two techniques has been evaluated in certain cohorts. The comparison of plasma cfDNA and tissue DNA NGS ina large Chinese cancer cohort is yet to be analyzed. Methods: We retrospectively reviewed HaploX's genomic sequencing data from July 2021 to December 2022 to assess the concordance between plasma cfDNA and cancer tissue DNA. A total of 589 advanced lung cancer patients with matched tissue and plasma sample were included in the study. Tissue and peripheral blood was collected at the same time during surgical operation or biopsy. Tissue DNA and plasma cfDNA were isolated and prepared for the NGS with commercially available kits. The positive percent agreement (PPA) between plasma cfDNA sequencing and tissue sequencing, the negative percent agreement (NPA), positive predictive value (PPV), negative predictive value (NPV), and overall percent agreement (OPA) were calculated and compared. Results: The positive percent agreement (PPA) of plasma cfDNA sequencing compared with tissue sequencing were 78% (EGFR, 82%; KRAS, 71%; HER2 mutation, 76%; BRAF V600E mutation, 75%; ALK fusion, 74%; RET fusion, 60%; ROS1 fusion,33.3%; MET exon14 skipping, 50%). EGFR mutations included EGFR p.L858R,19del, p.C797S, p.L861Q, p.T790M, KRAS mutations included p.G12X and p.Q61X.， HER2 mutations included gain-of-function mutations；NPA was 92.4%，PPV was 93.8%，NPV was 74.4%，OPA was 84%. Conclusions: The positive mutation detection consistency of plasma cfDNA sequencing and tissue sequencing in advanced lung cancer patients is 78%, indicating that liquid biopsy based on plasma cfDNA is reliable in most advanced lung cancer patients, but there are some limitations in gene fusion and MET exon 14 jumping detection. Liquid biopsy based on plasma cfDNA can meet most clinical genomic testing needs in general, especially when tissue is not available, but physicians need to be more cautious with negative results. Keywords: Liquid biopsy, Next-generation sequencing (NGS), Concordance Analysis, Lung Cancer.

Characteristic computed tomography features in mesenchymal-epithelial transition exon14 skipping-positive non-small cell lung cancer

BackgroundMesenchymal-epithelial transition exon14 (METex14) skipping is one of the therapeutic driver oncogene mutations in non-small cell lung cancer (NSCLC), and can be treated with tepotinib and capmatinib. There is only one report on computed tomography (CT) findings of METex14 skipping-positive NSCLC, which shows that the primary tumor tends to have a large mass in the upper lobe, and extrathoracic metastases are common. This study examined the CT findings of METex14 skipping-positive NSCLC, focusing on the features of the margins and internal structures.MethodsWe consecutively included patients with METex14 skipping-positive NSCLC who were diagnosed between January 2018 and December 2020 at four independent institutions. We retrospectively reviewed the patient demographics and CT findings for tumor margins (invasion into surrounding tissue, lobulation, pleural indentation, spicula, and ground-glass opacity) and internal structures (air bronchograms, cavitation and internal low-density area).ResultsFifteen patients with METex14 skipping-positive NSCLC were identified. Almost half of the patients were men (7/15; 46.7%), and their median age was 75.0 years. More than half were either current or former smokers (9/15; 60.0%). A vast majority of histological subtypes were adenocarcinoma (10/15; 66.7%), followed by pleomorphic carcinoma (3/15; 20.0%) and squamous cell carcinoma (2/15; 13.3%). With regard to CT findings, most primary tumors presented as masses larger than 30 mm (12/15; 80.0%) and were located in the upper lobes (12/15; 80.0%). Invasion into surrounding tissue and presence of internal low-density areas were observed in 60.0% (9/15) and 66.7% (10/15) of the primary tumors, respectively. Additionally, their frequencies increased to 72.7% (8/11) and 90.9% (10/11) in stage III/IV cases, respectively. In lymph node metastasis, internal low-density areas were observed in 8/10 cases (80.0%). Although these two CT features were rarely observed in distant metastases at diagnosis, they became apparent with progression of the metastatic tumor size.ConclusionsMETex14 skipping-positive NSCLC tumors tend to invade surrounding tissue and possess internal low-density areas. These CT findings might be characteristic of METex14 skipping-positive NSCLC.

A lung adenocarcinoma patient with co-mutations of MET and EGFR exon20 insertion responded to crizotinib

BackgroundTargeted therapy has revolutionized the treatment of patients with malignancies harboring mutations in driver genes and has brought a favorable survival benefit to the population with actionable oncogenic mutations. In recent years, the MET exon14 skipping mutation has been recognized as a potentially promising therapeutic target in non-small cell lung cancer (NSCLC). These changes are mutually exclusive with molecular drivers such as EGFR, KRAS, HER-2, BRAF, ALK and ROS1. The prevalence rate of coexisting MET exon 14 mutations and EGFR sensitive mutations (L858R, exon 19 deletions) in Chinese population was reported to be 0.2% (3/1590). However, the coexistence of MET exon 14 mutations with EGFR exon 20 insertion mutations has never been reported and the management of this subtype is not identified.Case presentationA 69-year-old male with a right lung adenocarcinoma (T4N2M0, IIIB) was confirmed to be positive for MET exon 14 skipping (c.3028_3028+1delGGinsTT, 44.4%), MET amplification (copy number 4.4), and EGFR exon 20 insertion (p. N771_H773dup, 22.1%) mutations. After the progression of one cycle of chemotherapy (Pemetrexed 0.8 g d1), the patient was subsequently accepted treatment with Crizotinib (250 mg twice a day) and achieved an important clinical remission for six months until the development of brain metastases. Then, he was submitted to a cycle of anti-programmed cell death-1 (PD-1) therapy after failure of Crizotinib and eventually acquired resistance despite of the high expression of programmed death ligand-1 (PD-L1) and tumor mutational burden (TMB) status.ConclusionThis case report provides treatment strategies for epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs)-untreated lung adenocarcinoma patients simultaneously carrying MET alterations and EGFR exon 20 insertion mutations. In addition, the signatures of PD-L1 or TMB expression were not the candidate for predicting the efficacy of immunotherapy in this context.

Abstract 78: Detection of gene fusions and exon skipping events in lung FFPE samples with Oncomine Precision Assay on Ion Torrent Genexus࣪ System

Abstract Introduction: Gene fusions and exon skipping events play an important oncogenic role in non-small cell lung cancer (NSCLC). Here we employed the Oncomine Precision Assay (OPA) for sequencing of 998 clinical research FFPE (Formalin-Fixed Paraffin-Embedded) lung samples using the Genexus࣪ integrated sequencing platform. The RNA assay strategy is aimed at providing a wide scope for studying known oncogenic fusions and exon skip variants, as well as a method for detection of novel fusion combinations and detection of fusions in a partner agnostic manner. We summarize the findings that include detected samples with oncogenic fusions in tyrosine kinase genes ALK, RET, ROS1 as well as MET exon14 skipping, and demonstrate the novel fusion detection capabilities of the panel. Methods: The Oncomine Precision Assay panel developed using the Ion AmpliSeq HD technology for use on both tissue and liquid biopsy samples to detect fusions with high sensitivity in low input RNA. The panel features 981 known fusion isoforms in 16 oncogenic drivers as well as assays for exon skipping and deletion in MET and EGFR. The amplicons are strategically designed around known fusion breakpoints and generate reads only if the variant is present. Results are assessed bioinformatically with a framework that includes detailed genomic annotations of the fusion breakpoint, and generates interpretable fusion calls and report in the Genxus࣪ software. In addition, the panel contains an algorithm for novel fusion detection, and 78 exon-junction amplicons for partner agnostic fusion detection in ALK, RET, NTRK1,2,3 and FGFR1,2,3 with an exon tiling expression imbalance assay. We used the Genexus࣪ instrument to sequence 998 unique lung FFPE samples and analyzed the results with the Genexus࣪ fusion analysis workflow. Results: A total of 998 unique lung FFPEs were sequenced, of which 906 (91%) resulted with >5000 aligned reads and at least 5 of the 7 RNA expression controls at the assay threshold. A total of 25 samples with ALK fusions were detected by either the targeted fusions or the expression imbalance assays. In addition, we detected 10 samples with RET fusions, 4 with FGFR3 fusions, 3 with ROS1 fusions, and additional single observations of NRG1, RSPO3 and FGFR1 fusions. Exon 14 skipping in MET was detected in 14 samples, of which we were able to detect a likely splice site variant SNV or indel at, or near the boundaries of exon 14, with the DNA assays of the panel. These variants were observed in a mutually exclusive set of 59 total samples (6.5% of the samples). Conclusions: We used the OPA panel for fusions and intragenic rearrangements that retains the simple workflow and fast turn-around time of previous Oncomine fusion, and demonstrated known and novel fusion detection as well exon skip variant detection capabilities in a research cohort of lung FFPE samples. For research use only. Not for use in diagnostic procedures. Citation Format: Amir Marcovitz, Jeoffrey Schageman, Jian Gu, Stephen Wunsch, David Chi, Paul D. williams, Scott P. Myrand, Fiona C. Hyland, Seth Sadis, Kelli S. Bramlett. Detection of gene fusions and exon skipping events in lung FFPE samples with Oncomine Precision Assay on Ion Torrent Genexus࣪ System [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 78.

A phase 1 dose-escalation study of the ABN401 (c-MET inhibitor) in patients with solid tumors.

3105 Background: MET is a proto-oncogene encoding a receptor tyrosine kinase c-MET for hepatocyte growth factor (HGF). Dysregulated MET signaling by MET exon14 skipping, MET gene amplification and c-MET overexpression in cancer plays a critical role in the development of primary oncogenesis, acquired drug resistance and metastasis. This is a first-in-human trial, phase 1 dose-escalation study of the highly selective MET kinase inhibitor, ABN401. ABN401was evaluated in subjects with advanced solid tumors in South Korea and Australia. Methods: Patients with advanced solid tumors were enrolled in escalating dose cohorts using an accelerated titration design. ABN401 was orally administered daily with 21-day cycle. The primary objective was to evaluate safety and tolerability to define dose-limiting toxicity (DLT), maximum tolerated dose (MTD) according to CTCAE v5. Secondary objectives included pharmacokinetic, recommended phase II dose (RP2D), and preliminary efficacy assessments. Tumor assessment was determined using Response Evaluation Criteria in Solid Tumors (RECIST 1.1). Results: Out of 28 screened patients, 16 patients with 6 different tumor types were treated with ABN401 at daily dose levels of 50, 100, 200, 400, 800 and 1200 mg, 15 patients were evaluated for DLT and one unevaluable. No DLT was observed in all 6 dose levels and the MTD has not been reached. No drug related grade ≥ 3 AEs were observed: only one drug-related SAE (transient peripheral edema) was reported. For treatment response, 5 patients with stable disease, and 2 with partial response were observed. These two patients with partial response had non-small cell lung cancer (NSCLC) with c-MET overexpression and had been treated with ABN401 for 10 and 18 months, respectively. Conclusions: ABN401 dosed up to 1200 mg QD was well tolerated with an acceptable safety profile and promising preliminary antitumor activity in patients with advanced solid tumors. The extension (pilot expansion) for additional efficacy assessment is under way at 800 mg daily dose with c-MET altered NSCLC patients in South Korea and Australia. In addition, a phase 2 expansion study is to start in the United States and South Korea. Clinical trial information: NCT04052971.

Driver coexistence characteristics of ALK-fusion in Chinese patients with lung cancer.

8530 Background: ALK fusions and other driver mutations are usually mutually exclusive. With the widespread application of genetic testing techniques, the coexistence of ALK fusions and other driver mutations could be detected, such as ALK fusion and EGFR driver mutations. However, there were no systematical studies about the coexistence of ALK fusions and other driver mutations. Here, we retrospectively investigated the coexistence of ALK fusions and other driver mutations. Methods: Samples with ALK fusions were extracted from a Chinese lung cancer cohort, which from OncoPanscan (Genetron Health) based sequencing of tissue. Driver mutations of EGFR, ROS1, RET, NTRK1/2/3, BRAF, MET, KRAS and ERBB2 could be detected. Results: In the cohort, 692 samples with ALK fusions contained the intact kinase domain. These samples could be classified into three forms: canonical fusions (only EML4 partner, n = 601), single non-canonical fusions (only non- EML4 partner, n = 51) and complex non-canonical fusions (two or more partners, n = 40). Among the 692 samples, only 20 samples (20/692, 2.89%) coexisted with other driver mutations, which indicated that the driver coexistence were rare. 70% (14/20) of driver coexistence were happened on canonical fusions ( EML4-ALK) samples. They were coexisting with EGFR L858R (3), EGFR L858R plus ROS1 fusion (1), EGFR L858R plus MET amplification (1), KRAS G12C/D/V (3), MET amplification (3), MET Exon14 skipping (1), ERBB2 amplification (1), and MET amplification plus ERBB2 amplification (1), respectively. 20% (4/20) of driver coexistence samples were single non-canonical ALK fusions coexisting with EGFR 19del (1) or 20ins (1) or L858R (1) or MET amplification (1). Another 10% (2/20) samples were complex non-canonical ALK fusions coexisting with EGFR G719S (1) or RET fusion (1). Most co-mutations have corresponding targeted inhibitors, maybe these patients can be treated by combined or sequential therapies. Among canonical fusions, single non-canonical fusions and complex non-canonical fusions of ALK, the frequency of the samples without coexistence of driver mutations was respectively 97.67% (587/601), 92.16% (47/51), 95.00% (38/40), without significant difference (P = 0.056). Maybe single/complex non-canonical fusions are also strong drivers as canonical fusions. Conclusions: In this cohort, very few of ALK fusion patients coexisted with other driver mutations. Among the co-existence samples, ALK fusion were mainly coexisting with the site mutations of EGFR and KRAS, amplifications of MET and ERBB2, fusions of ROS1 and RET. These samples maybe obtain more effective outcomes by combined or sequential therapies.

Fmr1 exon 14 skipping in late embryonic development of the rat forebrain

BackgroundFragile X syndrome, the major cause of inherited intellectual disability among men, is due to deficiency of the synaptic functional regulator FMR1 protein (FMRP), encoded by the FMRP translational regulator 1 (FMR1) gene. FMR1 alternative splicing produces distinct transcripts that may consequently impact FMRP functional roles. In transcripts without exon 14 the translational reading frame is shifted. For deepening current knowledge of the differential expression of Fmr1 exon 14 along the rat nervous system development, we conducted a descriptive study employing quantitative RT-PCR and BLAST of RNA-Seq datasets.ResultsWe observed in the rat forebrain progressive decline of total Fmr1 mRNA from E11 to P112 albeit an elevation on P3; and exon-14 skipping in E17–E20 with downregulation of the resulting mRNA. We tested if the reduced detection of messages without exon 14 could be explained by nonsense-mediated mRNA decay (NMD) vulnerability, but knocking down UPF1, a major component of this pathway, did not increase their quantities. Conversely, it significantly decreased FMR1 mRNA having exon 13 joined with either exon 14 or exon 15 site A.ConclusionsThe forebrain in the third embryonic week of the rat development is a period with significant skipping of Fmr1 exon 14. This alternative splicing event chronologically precedes a reduction of total Fmr1 mRNA, suggesting that it may be part of combinatorial mechanisms downregulating the gene’s expression in the late embryonic period. The decay of FMR1 mRNA without exon 14 should be mediated by a pathway different from NMD. Finally, we provide evidence of FMR1 mRNA stabilization by UPF1, likely depending on FMRP.

Clinicopathologic and Genomic Landscape of Non-Small Cell Lung Cancer Brain Metastases.

BackgroundIn patients with non-small cell lung cancer (NSCLC), 10%-40% will eventually develop brain metastases. We present the clinicopathologic, genomic, and biomarker landscape of a large cohort of NSCLC brain metastases (NSCLC-BM) samples.Materials and MethodsWe retrospectively analyzed 3035 NSCLC-BM tested with comprehensive genomic profiling (CGP) during routine clinical care. In addition, we compared the NSCLC-BM to a separate cohort of 7277 primary NSCLC (pNSCLC) specimens. Finally, we present data on 67 paired patients with NSCLC-BM and pNSCLC.ResultsComprehensive genomic profiling analysis of the 3035 NSCLC-BMs found that the most frequent genomic alterations (GAs) were in the TP53, KRAS, CDKN2A, STK11, CDKN2B, EGFR, NKX2-1, RB1, MYC, and KEAP1 genes. In the NSCLC-BM cohort, there were significantly higher rates of several targetable GAs compared with pNSCLC, including ALK fusions, KRAS G12C mutations, and MET amplifications; and decreased frequency of MET exon14 skipping mutations (all P < .05). In the subset of NSCLC-BM (n = 1063) where concurrent PD-L1 immunohistochemistry (IHC) was performed, 54.7% of the patients with NSCLC-BM were eligible for pembrolizumab based on PD-L1 IHC (TPS ≥ 1), and 56.9% were eligible for pembrolizumab based on TMB-High status. In addition, in a series 67 paired pNSCLC and NSCLC-BM samples, 85.1% (57/67) had at least one additional GA discovered in the NSCLC-BM sample when compared with the pNSCLC sample.ConclusionsHerein, we defined the clinicopathologic, genomic, and biomarker landscape of a large cohort of patients with NSCLC-BM which can help inform study design of future clinical studies for patients with NSCLC with BM. In certain clinical situations, metastatic NSCLC brain tissue or cerebral spinal fluid specimens may be needed to fully optimize personalized treatment.

2MO Final OS results and subgroup analysis of savolitinib in patients with MET exon 14 skipping mutations (METex14+) NSCLC

Savolitinib, a highly selective MET tyrosine kinase inhibitor, has shown promising activity and tolerable safety in patients (pts) with pulmonary sarcomatoid carcinoma (PSC) and other NSCLCs harboring MET exon14 skipping mutations (METex14+) (Lu, 2021). Here we report the long-term overall survival (OS), exposure and subgroup analysis results.

P60.09 Real-World Readiness of US Laboratories to Test Metastatic NSCLC Patients for Rare Actionable Genomic Variants by NGS

Various rare driver genomic variants have been investigated in metastatic non-squamous non-small cell lung cancer (mNSCLC). Individually they are considered rare (<3% prevalence), however, studies have shown collectively they represent up to 14.5% of the mNSCLC mutation profile. While multiple targeted therapies for rare variants such as NTRK fusions, MET exon14 skipping and RET fusions have been approved in the US for 1st line therapy in mNSCLC, other variants such as HER2 exon20ins, EGFR exon20ins, and FGFR-alterations are also gaining traction.

1258P MET exon14 skipping in non-small cell lung cancer: Clinicopathological characteristics, treatments, and efficacy of crizotinib according to functional analysis: AFonMET - GFPC study

MET targeted tyrosine kinase inhibitors (TKI) demonstrated efficacy in advanced non-small cell lung cancer (aNSCLC) with METexon 14 skipping mutation (METexon14). Different variants of this mutation exist. Some do not affect the splice sites and may result in incomplete exon 14 skipping. This could explain a different anti-tumor activity of TKI.Functional analysis could specify if the exon skipping is effective.

RNA sequencing effectively identifies gene fusions undetected by DNA sequencing in lung adenocarcinomas.

3052 Background: Next-generation sequencing of DNA, which can provide valid information for clinical therapeutic decision-making, has been widely used in the management of lung cancer especially adenocarcinoma. However, due to its technical limitations for detecting certain alterations such as gene rearrangement, the DNA-based sequencing (DNA-seq) may miss the actionable alteration in some cases, who would have benefited from targeted therapy. The study aimed to evaluate the capability of RNA sequencing (RNA-seq) in identifying DNA-seq undetectable gene alterations in lung adenocarcinomas. Methods: A total of 219 lung adenocarcinomas, which had no driver alteration detected by DNA-seq (OncoScreen Plus, Burning Rock Biotech) and had a max AF ≥5%, underwent capture-based RNA-seq using a custom panel (OncoRNA, Burning Rock Biotech) spanning full transcripts of 115 genes commonly involved in cancer genomic rearrangements. Furthermore, an independent cohort of 100 DNA-seq driver–negative lung adenocarcinomas were also subjected to RNA-seq with the same panel. Results: In the discovery cohort, 166/219 samples (75.8%) generated qualified RNA-seq data for subsequent analyses. RNA-seq identified 44 previously undetected alterations (26.5%), including 40 gene fusions (24.1%), 1 MET exon14 skipping variant ( METex14, 0.6%) and 3 other alternative splicing variants (1.8%). Among them, 14 (8.4%) were potential actionable alterations, consisting of METex14 and in-frame fusions containing functional domain of the driver gene (4 ROS1 fusions, 3 BRAF fusions, 2 NRG1 fusions, 2 EGFR fusions, 1 ALK fusion and 1 MET fusion). In the validation cohort, 69/100 samples (69.0%) generated qualified data. RNA-seq identified 22 DNA-seq undetected alterations (31.9%), with 7 of them being potential actionable fusions (10.1%). ROS1 fusion remained as the most common actionable alteration (n = 3), followed by ALK fusion (n = 2), EGFR fusion (n = 1) and MET fusion (n = 1). Further analyses of the two datasets revealed that lacking sufficient coverage spanning the rearrangement breakpoint in the DNA-seq panel mainly accounted for the failure of DNA-seq on detecting these fusions. This can be improved by increasing the corresponding probe coverage in the DNA-seq panel. In addition, complex genomic rearrangement at DNA level and the presence of repetitive sequence in the intronic region spanning or adjacent to the breakpoint might lead to missed calling of canonical fusions by DNA-seq. Conclusions: Targeted RNA-seq can effectively identify genomic rearrangements that are undetectable by DNA-seq and provide lung adenocarcinoma patients with more opportunities for targeted therapy. Therefore, it should be recommended for all patients, in whom DNA-seq fails to detect driver alteration.

Incidence and PD-L1 Expression of MET 14 Skipping in Chinese Population: A Non-Selective NSCLC Cohort Study Using RNA-Based Sequencing.

BackgroundMesenchymal–epithelial transition (MET) exon14 skipping mutations represent a clinically unique molecular subtype of NSCLC. The prevalence rates of MET exon 14 skipping in lung adenocarcinoma (ADC) range from 0.9% to 4.0% in Asian populations. Since some somatic variants that do not encompass the MET exon 14 splice sites might also induce MET exon 14 skipping, the RNA-based sequencing is speculated as the most accurate method for detecting exon 14 skipping.Patients and MethodsA total of 951 NSCLC patients from two hospitals were enrolled in this study. MET exon14 skipping was detected using RNA-based next-generation sequencing (NGS). Also, immunohistochemistry (IHC) was performed in 405 samples simultaneously.ResultsThe overall estimated prevalence of MET exon 14 skipping was approximately 1.8% in ADCs and 1.7% in NSCLCs. The detection rate of MET exon 14 skipping from surgical resection specimen was 2.3% in NSCLCs and 2.0% in ADCs. The MET exon 14 skipping was identified in 6.6% of EGFR/KRAS/ALK/ROS1/RET-negative ADCs. Additionally, PD-L1 was found to be highly expressed in NSCLC patients harboring MET exon 14 skipping (P<0.01).ConclusionThe prevalence of MET exon14 skipping in lung ADCs in the East Asian population was similar to that of the Western population as assessed by RNA-based NGS. The NSCLC patients with MET exon 14 skipping were older than those with other oncogenic driver mutations, such as EGFR, ALK, and ROS1. In addition, PD-L1 was highly expressed in NSCLC patients with MET exon 14 skipping.

Analysis of multigene detection in patients with advanced lung adenocarcinoma using cytological specimens

ObjectiveTo investigate the mutation status and clinical characteristics of multigene detection in advanced lung adenocarcinoma using cytological specimens. Materials and Methods137 advanced lung adenocarcinoma patients with 10 driver genes detection in the Fourth Hospital Hebei Medical University from January 2019 to November 2019 was analysized. 137 cytological specimens including fine-needle aspiration specimens and maligant serous cavity effusion (pleural effusion, peritoneal and pericardial effusion). Ten driver mutations of EGFR, ALK, ROS1, BRAF, KRAS, NRAS, HER2, RET, PIK3CA and MET were detected by the amplification refractory mutation system (ARMS). Meanwhile, 90 of 137 patients were detected with biopsies for parallel gene detection. Results78.10 % (107/137) of patients with advanced lung adenocarcinoma harbored at least one of 10 driver mutations. The three main mutations were EGFR (69.16 %, 74/137), ALK (6.57 %, 9/137)and ROS1 (3.65 %, 5/137) mutations. Besides, we found 6 cases including two concomitant mutations: EGFR Exon19 del/HER2 (1/137), EGFR Exon21 L858R/PIK3CA (2/137), EGFR Exon21 L858R/RET (1/137), and ALK/KRAS (2/137). Among 137 patients, women aged 64 or older were more likely to have the mutations (P < 0.05). Female patients (P = 0.003) older or equal to 64 years (P = 0.015) with non-smoking habbit (P = 0.027) were more detected with EGFR mutations, while ALK was more detectable in patients yonger than 64 years. Parallel analysis showed that rates of single EGFR, ALK, ROS1, RET, KRAS, NRAS, HER2, MET mutations and concomitant different mutations were not significantly different between cytological specimens and matched histological specimens. ConclusionsIn the study, cytological specimens and biopsy samples have a very high coincidence rate of gene detection. EGFR, ALK and ROS1 mutations were the main driver mutations in patients with advanced lung adenocarcinoma.We speculate that EGFR and ALK are more prone to concomitant mutations respectively and the treatment of advanced lung adenocarcinoma patients with concomitant mutations deserves further study. The rate of KRAS, NRAS, BRAF, PIK3CA, RET and MET exon14 skipping mutation were low but may had a significant impact on the targeted therapy of patients with advanced lung adenocarcinoma.